U.S. patent application number 13/521695 was filed with the patent office on 2013-03-07 for process for the preparation of a biomass comprising plantaricin and uses thereof in medical field.
This patent application is currently assigned to GIULIANI S.p.A.. The applicant listed for this patent is Anna Benedusi, Maria Calasso, Maria De Angelis, Raffaella Di Cagno, Giammaria Giuliani, Marco Gobbetti. Invention is credited to Anna Benedusi, Maria Calasso, Maria De Angelis, Raffaella Di Cagno, Giammaria Giuliani, Marco Gobbetti.
Application Number | 20130059790 13/521695 |
Document ID | / |
Family ID | 42238665 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130059790 |
Kind Code |
A1 |
Giuliani; Giammaria ; et
al. |
March 7, 2013 |
PROCESS FOR THE PREPARATION OF A BIOMASS COMPRISING PLANTARICIN AND
USES THEREOF IN MEDICAL FIELD
Abstract
The present invention concerns a process for preparation of a
biomass comprising one or a more plantaricins A, N or K in
association with the lactic acid bacteria used for the preparation
and uses thereof in order to stimulate the barrier function of
intestinal cells or human epidermal keratinocytes.
Inventors: |
Giuliani; Giammaria;
(Milano, IT) ; Benedusi; Anna; (Milano, IT)
; Gobbetti; Marco; (Bari, IT) ; Di Cagno;
Raffaella; (Milano, IT) ; De Angelis; Maria;
(Bari, IT) ; Calasso; Maria; (Bari, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Giuliani; Giammaria
Benedusi; Anna
Gobbetti; Marco
Di Cagno; Raffaella
De Angelis; Maria
Calasso; Maria |
Milano
Milano
Bari
Milano
Bari
Bari |
|
IT
IT
IT
IT
IT
IT |
|
|
Assignee: |
GIULIANI S.p.A.
Milano
IT
|
Family ID: |
42238665 |
Appl. No.: |
13/521695 |
Filed: |
January 4, 2011 |
PCT Filed: |
January 4, 2011 |
PCT NO: |
PCT/IT11/00003 |
371 Date: |
November 16, 2012 |
Current U.S.
Class: |
514/18.6 ;
435/252.9; 435/71.3; 514/21.3 |
Current CPC
Class: |
C12P 39/00 20130101;
C07K 14/335 20130101; C12R 1/25 20130101; A61P 43/00 20180101; A61P
17/00 20180101; C12N 1/20 20130101; A61K 38/164 20130101; A61K
35/74 20130101; A61P 1/04 20180101; C12R 1/225 20130101; A61P 17/02
20180101 |
Class at
Publication: |
514/18.6 ;
435/71.3; 435/252.9; 514/21.3 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 17/02 20060101 A61P017/02; A61P 17/00 20060101
A61P017/00; C12P 21/00 20060101 C12P021/00; C12N 1/20 20060101
C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 2010 |
IT |
RM2010A000004 |
Claims
1. Biotechnological process for the synthesis of a biomass
comprising or consisting of at least one plantaricin selected from
plantaricins A, K or N, or mixtures thereof and Lactobacillus
plantarum DSM 23213 and Lactobacillus sanfranciscensis DSM 23214
lactic acid bacteria or for the preparation of one or more
plantaricins selected from plantaricins A, K or N or mixtures
thereof, said process comprising or consisting of the following
steps: a) culture propagation of Lactobacillus plantarum DSM 23213
and Lactobacillus sanfranciscensis DSM 23214 lactic acid bacteria;
b) co-inoculation of a substrate selected from the group consisting
of CDM, WFH, grape must, milk serum or fruit and vegetable product
extracts, with aqueous suspension of lactic acid bacteria as
defined in step a); c) incubation; and, optionally, d)
centrifugation of the culture broth in order to remove the lactic
acid bacteria cells.
2. Process according to claim 1, wherein the cell density of
suspension from step a) is about 9.0 Log ufc/ml for each lactic
acid bacteria species and it is added to the substrate in a
percentage ranging from 1 to 4% respect to the substrate
volume.
3. Process according to claim 1, wherein the incubation is carried
out at a temperature from 30 to 37.degree. C., preferably
30.degree. C., for 18-24 hours, preferably 18 hours.
4. Process according to claim 1, wherein the centrifugation is
carried out at 10.000.times.rpm for 15 min at 4.degree. C.
5. Process according to claim 1, further comprising the step e) for
dehydration of the supernatant obtained in step d) by means drying
or freeze-drying.
6. Biomass obtainable or obtained by means of the process as
defined in claim 1, said biomass comprising or consisting of at
least one plantaricin selected from plantaricins A, K or N or
mixtures thereof and Lactobacillus plantarum DSM 23213 and
Lactobacillus sanfranciscensis DSM 23214 lactic acid bacteria.
7. Pharmaceutical or cosmetic composition comprising or consisting
of the biomass as defined in claim 6, as an active principle, in
association with one or more pharmaceutically acceptable excipients
and/or adjuvants.
8. A method of increasing the barrier function of epidermis or
intestinal wall of a subject, comprising administering to the
subject the biomass of claim 6.
9. A method of wound healing in a subject, comprising administering
to the subject the biomass of claim 6.
10. Lactobacillus plantarum DSM 23213 or Lactobacillus
sanfranciscensis DSM 23214 lactic acid bacteria or mixture
thereof.
11. A method of increasing the barrier function of epidermis of a
subject, comprising administering to the subject one or more
plantaricins selected from plantaricins A, K or N, preferably
A.
12. A method of would healing in a subject, comprising
administering to the subject one or more plantaricins selected from
plantaricins A, K or N, preferably A.
13. A method of increasing the barrier function of epidermis or
intestinal wall, comprising using the composition of claim 7.
14. A method of wound healing, comprising using the composition of
claim 7.
Description
[0001] The present invention concerns a process for preparation of
biomass comprising plantaricin and uses thereof in medical field.
In particular, the present invention concerns a process for
preparation of plantaricin A, N or K or mixtures thereof or a
biomass containing one or more of above mentioned plantaricins in
association with lactic acid bacteria used for preparation and uses
thereof in order to stimulate the barrier function of intestinal
cells or human epidermal keratinocytes.
[0002] It is known that epidermis and intestinal wall carry out a
barrier function against external environment and harmful agents
therein. Epidermis and internal wall barrier function can be
weakened due to various factors. One of the conditions more
frequently resulting in alterations of epidermis barrier function
is the exposure to variously originating environmental factors.
Under normal conditions anyway the epidermis is able to adapt to
possible exogenous injuries through an adaptation resulting in a
"steady state" achievement and adequate tolerance grade. Epidermis
continuous aggression by irritating detergents and chemical
substances (organic solvents, soaps, detergent solutions), for
example, can damage not only the lipid component of epidermis
surface film, but also corneous layer intercellular one resulting
in barrier function degradation.
[0003] This results in low grade dermatitis characterized by an
increase of water trans-epidermal loss, minor desquamation and
elasticity loss of corneous layer, possibly followed by the
formation of small continuous surface solutions. This mild
irritating condition, often sub-clinical, triggers restoration
processes that, through an increase of lipid synthesis and
stimulation of basal keratinocyte proliferation activity allows new
equilibrium and barrier function restoration to be achieved.
[0004] When dermal barrier is no more suitably to carry out
adequately intrinsic defensive functions, the risk for onset of
inflammatory cutaneous pathologies, triggered by cytokine release,
resulting in the production in situ of flogogenic mediators and
free radicals, increases. The latter, in addition to generation by
oxidative mechanism direct DNA and protein injuries, can cause the
peroxidation of dermal cellular membranes.
[0005] Although epidermis does not contain blood vessels, anyway
contains a small water amount indispensable for physiological
equilibrium and corneous tissue integrity. Water from dermal
capillaries flows through the dermal-epidermal junction and channel
among corneocyte rows up to levels similar to most tissues, i.e.
about 60-70% in the deep zones (compact corneous), and 10-35% in
more superficial regions (disjunct corneous). The fact that, also
in more external part, in contact with air, the epidermis is able
to maintain a moisture reservoir is based on two particular
functions: the barrier activity preventing the evaporation of fluid
and the global hydrophilic activity of corneous layer (water
holding capacity), resulting from the presence of high hygroscopic
particles. Experiments based on progressive stripping of thinnest
corneocyte layers of disjunct corneous layer, and hydrolipid
overlapping film, demonstrate that barrier function is not
remarkably affected, while, on the other hand, the same is
gradually degraded by the removal of deeper layers of underlying
compact corneous layers, whose cemented wall structure, with
corneocyte bricks consisting of flaggrin compacted keratin clusters
and rigid protein envelope is well known. By electron microscopy it
has been shown that corneocytes are tightly sealed by modified
desmosomial plates (corneosomes) and are embedded within a lipid
adhesive known as intercorneocyte concrete, generating a film of
flexible and nearly impenetrable barrier. Stressful conditions
possibly affect acutely or chronically the cutaneous barrier and
against the same the body generates a series of homeostatic
mechanisms resulting in the capacity of the epidermis to monitor
and restore the efficiency when failure thereof occurs
independently on the injurious acute noxa origin. The epidermis
responds to chronic cutaneous stresses, induced by an extended
exposure to low moisture air, as typically in dry climates occurs,
through compensative adaptive phenomena resulting in proliferation
increase of basal cells and consequent thickness increase of
corneous layer and epidermis as a whole. Also the production and
exocytosis of Odland bodies and intercorneocyte lipids (maintaining
usual composition) increase so as the epidermis can retain water
resources in a better way. In any case moisture deficiency results
in a phase change as to the liquid-lipid stratification of the
intercorneocyte concrete with subsequent crystallization. As a
result lesser plasticity and rigidity as to tractions induced by
the muscle-articular movements and extrinsic dynamic stimuli
occurs, thus easy microlesions are generated. Another implication
of similar adaptive responses consists of hyperkeratosis: the
proliferation layer increase is not counterbalanced by a
corresponding higher exfoliation rate of corneocytes that, rather
than to be scaled, remain embedded in corneous massive clusters and
are detached only as large scales. Moreover, chronic environmental
stresses activate a cytokine cascade with phlogosis histological
aspects in association with hypertrophy and degranulation of derma
mastocytes. This can explain the exacerbation of inflammatory
dermatopathologies as observed in winter. Every acute injury
against epidermis barrier induces a cytokine response by involved
cutaneous cells, the effects thereof are reflected on keratinocytes
and underlying derma, resulting in important morphologic and
functional consequences aiming to the restoration of the barrier
function, epidermal surface texturing, and, above all, stimulation
of fibroblasts activity on which the improvement of derma
compactness and turgor is based on.
[0006] Actually the methods used in order to restore the barrier
function are based on the use of restoring and hydroregulating
substances suitable to obviate to insufficient cutaneous defense
resulting from the alteration of the barrier function. These
substances are compounds with re-hydrating and restructuring
activity. Many dermocosmetic products allow the cutaneous
re-hydration and according to restoring function thereof, favour
cutaneous regeneration. However the same act through a simple
"cosmetic masking" of a rough surface and do not possess any true
therapeutic effect, that would have to exert through a repairing
and regenerative activity aiming to the restoration of cutaneous
morphofunctional integrity.
[0007] It has been recently outlined that bacteria are suitable to
release and detect signal molecules as a response to environmental
condition modifications, including variations of cellular density
thereof and/or number of other microbial cell species occurring
within an ecosystem (Sturme et al., 2007. Making sense of quorum
sensing in lactobacilli: a special focus on Lactobacillus plantarum
WCFA1. Microbilogy 153: 3939-3947). As to lactic acid bacteria,
these responses, occurring according to a "quorum sensing" (OS)
mechanism, include signal molecules named type 2 (Al-2, mainly
furanones derivatives) auto-inducers, synthesized using LuxS enzyme
activity (Miller e Bassler, 2003. LuxS quorum sensing: more than
just a numbers game. Curr Opin Microbiol 6: 191-197), or signal
molecules named peptide pheromone or peptide auto-inducer
(AIP)(Nakajama et al., 2001. Gelatinase biosynthesis-activating
pheromone: a peptide lactone that mediates a quorum sensing in
Enterococcus faecalis. Mol Microbiol 41: 145-154). It has been
recently proved that L. plantarum WCFS1 genome contains high number
of genes encoding for AlP peptides, together with other genes
encoding for other functions involved in "quorum sensing"
mechanisms (Sturme et al., 2007. Making sense of quorum sensing in
lactobacilli: a special focus on Lactobacillus plantarum WCFA1.
Microbilogy 153: 3939-3947). Some studies have proved that the
system aiming to the synthesis of plantaricin type peptide
pheromone is involved in intra-species cellular communication
mechanisms. In this case the peptide pheromone is used as a means
to measure the cell density of molecule synthesizing species (Diep
et al., 1994. The gene encoding plantaricin A, a bacteriocin from
Lactobacillus plantarum C11, is located on the same transcription
unit as an agr-like regulatory system. Appl Environ Microbiol
60:160-166). Other studies have also proved that plantaricin type
peptide pheromones can be involved in mechanisms of inter-species
cell communication. Particularly, the presence of competitive
microorganisms can activate the regulating system involved in
mechanisms of microbial antagonism (Maldonado et al., 2004.
Production of plantaricin NC8 by Lactobacillus plantarum NC8 is
induced in the presence of different types of Gram-positive
bacteria. Arch Microbiol 181: 8-16). In the presence of other
microbial species at cell high density, the peptide pheromone
favours a cascade series of phosphorylation reactions involving
metabolic regulation complex phenomena resulting in the synthesis
of signal molecules specifically acting as bacteriocin type
antimicrobial compounds (for example plantaricins A, K and N)
(Hauge et al., 1998. Plantaricin A is an ampkiphilic alpha-helical
bacteriocin-like pheromone which exerts antimicrobial and pheromone
activities through different mechanisms. Biochemistry
37:16026-16032).
[0008] Although the mechanism of cell communication among
prokaryotic and eukaryotic cells has been partially elucidated,
very limited literature exists as to interactions among signal
molecules involved in "quorum sensing" mechanisms of bacteria (for
example. peptide pheromones) and cells of human intestinal mucosa.
The unique example is CSF pentapeptide, synthesized by Bacillus
subtilis probiotic microorganism, as a molecule involved in
competence and sporulation phenomena (Fujija et al., 2007. The
Bacillus subtilis quorum-sensing molecule CSF contributes to
intestinal homeostasis via OCTN2, a host cell membrane transporter.
Cell Host Microbe 1:299-308). It has been demonstrated that said
pentapeptide is suitable to induce p38 MAP kinase, B kinase (Akt)
and cryotolerance, thus favouring the prevention of oxidative
damage at intestinal level and reinforcing barrier function.
Further at current state of the art no publication or patent
focused on the effect of signal molecules involved in mechanisms of
bacteria cellular communication with the respect to human epidermis
exists.
[0009] The Authors of the present invention now have discovered
that plantaricin, particularly plantaricin A, exerts a positive
effect on barrier function of keratinocytes in human epidermis and
intestinal cells as well.
[0010] Studies about processes for plantaricin preparation using
bacteria mono-cultures (intra-species) are known. However, the
culture of the interest microorganisms is carried out in complex
and too expensive culture media to be scaled up at industrial level
for the preparation of signal molecules for therapeutic
purpose.
[0011] The Authors of the present invention now have developed a
process for the preparation of plantaricin using a co-culture of
two specific lactic acid bacteria suitable to obtain a higher yield
than those obtained according to known art. Particularly the
cultivation of L. plantarum DC400 (deposited at DSMZ on 21 Dec.
2009 with number DSM 23213) as a co-culture with L. rossiae
DPPMA174 (deposited at DSMZ on 21 Dec. 2009 with number DSM 23214)
is suitable to activate the synthesis of plantaricin type peptide
pheromone (particularly plantaricin A) obtaining concentrations
about 50 fold higher than in the presence of L. plantarum DC400
monoculture (DSM 23213). Further it has been proved that L.
plantarum DC400 (DSM 23213) culture with other species of lactic
acid bacteria, also isolated by "natural sourdough", is not
suitable to stimulate the synthesis of peptide pheromone as when in
association with L. rossiae. Another important aspect of the
process according to the present invention is that the synthesis of
plantaricin A is viable not only in culture media usually employed
for lactic acid bacteria laboratory culture, but also on grape
must, milk serum and aqueous extracts from fruit and vegetable
products.
[0012] According to known art, no publication or patent disclosed
the plantaricin A type (PlnA) synthesis as a response mechanism to
co-culture of two lactic acid bacteria (for example. L. plantarum
and L. sanfranciscensis) occurring in the same alimentary
ecosystem, as "natural sourdough" used for the production of baked
leavened products. Further according to literature, it has not been
reported an increment of plantaricin A synthesis in co-culture
(inter-species) with respect to mono-culture conditions
(intra-species). In addition, as above reported, according to known
processes very expensive and complex culture media are used.
[0013] Moreover, it has been surprisingly discovered that the
biomass obtained according to the process of the invention
comprising one or more plantaricins in association with L.
plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214)
lactic acid bacteria exerts an higher effectiveness than
plantaricin alone in enhancing the barrier function at level of
epidermis or intestinal wall.
[0014] The lactic acid bacteria according to the present invention
belong to the Lactobacillus species and have been isolated from
"natural sourdoughs" for typical bread production in South
Italy.
[0015] A biotechnological protocol involving the co-culture of said
two bacteria in CDM (Chemically Defined Medium), WFH (Wheat Flour
Hydrolyzate) (Gobbetti, 1998. The sourdough microflora:
interactions of lactic acid bacteria and sourdoughs. Trends Food
Sci Technol 9:267-274), grape must (diluted at 1% of soluble
carbohydrates, added with 0.5% of maltose and 0.5% of sourdough
extract, pH 5.6), milk serum (added with 0.5% of maltose and 0.5%
of sourdough extract, pH 5.6) or aqueous extracts of vegetable and
fruit products (added with 0.5% of maltose and 0.5% of sourdough
extract, pH 5.6) for 18-24 hours at 30-37.degree. C. has been
standardized and optimized. At the end of the culture, the cells
can be removed or not from the broth-culture by centrifugation,
then the supernatant is subjected to a dehydrating process by
drying or freeze-drying.
[0016] A scheme of the biotechnological protocol for the
formulation of the preparation based on plantaricin A is described
below.
##STR00001##
[0017] When a culture of L. plantarum DC400 (DSM 23213) and L.
rossiae DPPMA174 (DSM 23214) under co-culture conditions on any of
above said substrates is carried out the synthesis of plantaricin A
at concentration from 2.5 to 4.0 .mu.g/mL has been detected. Under
mono-culture conditions, the concentration of plantaricin A
synthesized by L. plantarum DC400 (DSM 23213) is about 0.06
.mu.g/mL. Under co-culture conditions with other lactic acid
bacteria species (for example Pediococcus pentosaceus,
Lactobacillus pentosus, Lactobacillus brevis, Lactobacillus
rossiae, Lactobacillus rhamnosus) the synthesis of plantaricin A is
remarkably lower. Under co-culture conditions with L. rossiae
DPPMA174 (DSM 23214) the synthesis of other peptide pheromones, as
plantaricin type K and N has been detected, although at lower
concentrations than plantaricin A, and particularly in the range
from 0.02 to 0.06 .mu.g/ml. According to one possible formulation,
the application of 2.5 .mu.g/ml of plantaricin A stimulated the
barrier functions as proved using a reconstructed epidermis model
(SkinEthic.RTM.) and Transepitheliall Electric Resistance (TEER)
assay. Similar results have been obtained using Caco-2/TC7
intestinal cells suitable to reproduce the intestinal mucosa
[0018] According to complementary analyses carried out using
microbiological, chromatographic techniques and in vitro and
ex-vivo assays on cell cultures, the co-culture of L. plantarum
DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214), never used
in prior studies, according to the present invention, allows: (i)
the synthesis of signal molecules involved in inter-species cell
communication mechanisms at a concentration not detectable in the
presence of other lactic acid bacteria associations within a single
ecosystem; (ii) the synthesis of plantaricin A, and other
plantaricins (K and N), also using low cost substrates; and (iii) a
protective effect enhancing the barrier function at epidermis and
intestinal cell level, thus demonstrating that signal molecules
synthesized by prokaryotic cells are detected also by eukaryotic
cells.
[0019] It is therefore a specific object of the present invention a
biotechnological process for the synthesis of a biomass comprising
or consisting of at least one plantaricin selected from
plantaricins A, K or N, preferably A, or mixtures thereof and
Lactobacillus plantarum DSM 23213 and Lactobacillus rossiae DSM
23214 lactic acid bacteria or for the preparation of one or more
plantaricins selected from plantaricins A, K or N, preferably A, or
mixtures thereof, said process comprising or consisting of the
following steps:
a) culture propagation of Lactobacillus plantarum DSM 23213 and
Lactobacillus rossiae DSM 23214 lactic acid bacteria; b)
co-inoculation of a substrate selected from the group consisting of
CDM, WFH, grape must, milk serum or fruit and vegetable product
extracts, with aqueous suspension of lactic acid bacteria as
defined in step a); c) incubation; and, optionally, d)
centrifugation of the culture broth in order to remove the lactic
acid bacteria cells.
[0020] Particularly, the cell density of suspension from step a)
can be about 9.0 Log ufc/ml for each lactic acid bacteria species
and it is added to the substrate at percentage ranging from 1 to 4%
based on the substrate volume. The incubation step can be carried
out at a temperature from 30 to 37.degree. C., preferably
30.degree. C., for 18-24 hours, preferably 18 hours, while the
centrifugation can be carried out at 10000.times.g for 15 min at
4.degree. C.
[0021] The process according to the invention can further comprise
a step e) for dehydration of the supernatant obtained from step d)
by drying or freeze-drying.
[0022] It is a further object of the present invention a biomass
obtainable or obtained according to above said process, comprising
or consisting of at least one plantaricin selected from
plantaricins A, K or N, preferably A, or mixtures thereof and
Lactobacillus plantarum DSM 23213 e Lactobacillus rossiae DSM 23214
lactic acid bacteria.
[0023] The present invention further concerns a pharmaceutical or
cosmetic composition comprising or consisting of the biomass as
above defined, as an active principle, in association with one or
more pharmaceutically acceptable excipients and/or adjuvants.
[0024] A particular aspect of the present invention further refers
to the use of the biomass or composition as above defined for the
preparation of a medicament in order to increase the barrier
function of epidermis or intestinal wall or for wound healing.
[0025] Lactobacillus plantarum DSM 23213 or Lactobacillus rossiae
DSM 23214 lactic acid bacteria or mixtures thereof are a further
object of the present invention.
[0026] Further the present invention refers to the use of one or
more plantaricins selected from plantaricins A, K or N, preferably
A, for the preparation of a medicament in order to increase the
barrier function of epidermis or intestinal wall or for wound
healing.
[0027] The present invention now will be described by an
illustrative but not a limitative way, according to preferred
embodiments thereof, particularly with reference to the enclosed
drawings.
[0028] FIG. 1a shows results of electrospray-ionization (ESI) ion
trap MS (nano-ESI/MS-MS) coupled Multidimensional HPLC (MDLC)
analysis of free cell supernatant from broth-culture obtained by
co-culture of Lactobacillus plantarum DC400 (DSM 23213) and
Lactobacillus rossiae DPPMA174 (DSM 23214). FIG. 1b shows the
chromatogram obtained from real chromatogram of FIG. 1a based on
the specific acquisition time with m/z ratios relative to the
plantaricin A. FIG. 1c shows MS/MS spectrum detecting species based
on 1493.7 m/z ratio as observed in chromatogram of FIG. 1a.
[0029] FIG. 2 shows concentration of plantaricin A synthesized from
Lactobacillus plantarum DC400 (DC400), L. plantarum DPPMA 20
(DPPMA20), Lactobacillus pentoses 12H5 (12H5), Lactobacillus
rossiae DPPMA174 (DPPMA174) and Pediococcus pentosaceus
2.times.A.English Pound. (2.times.A3) mono-cultures and L.
plantarum DC400 with L. plantarum DPPMA20 (DC400-DPPMA20), L.
pentoses 12H5 (DC400-12H5); L. rossiae DPPMA174 (DC400-DPPMA174) or
P. pentosaceus 2.times.A3 (DC400-2.times.A3) co-cultures. Data is
the average of three triplicate experiments.
[0030] FIG. 3 shows growth kinetics of Lactobacillus rossiae
DPPMA174. Mono-culture ( ); co-culture with Lactobacillus plantarum
DC400 (.largecircle.); mono-culture with purified plantaricin A
(2.5 .mu.g/ml) (.DELTA.); and mono-culture with chemically
synthesized plantaricin A (2.5 .mu.g/ml) (.tangle-solidup.).
Purified plantaricin A corresponds to that synthesized by
co-culture of L. plantarum DC400 and L. rossiae DPPMA174. Data is
the average of three triplicate experiments.
[0031] FIG. 4 shows portions of gel relating to electrophoretic
two-dimensional analysis of Lactobacillus rossiae DPPMA174
expressed proteins after 18 hour culture. Panel A, mono-culture;
panel B, co-culture with Lactobacillus plantarum DC400; and panel
C, mono-culture in the presence of purified plantaricin A (2.5
.mu.g/ml). Oval or triangle marked numbers refer to proteins
displaying an expression level increase or decrease in the culture
with L. plantarum DC400 or purified plantaricin A. Rhomb or double
triangle marked numbers refer to proteins displaying an expression
level increase or decrease only in the co-culture with L. plantarum
DC400.
[0032] FIG. 5 shows Transepithelial Electric Resistance (TEER)
(Ohms.times.cm.sup.2) of reconstructed epidermis (SkinEthic.RTM.)
after exposure for 0 and 24 hours to PBS buffer, plantaricin A (2.5
.mu.g/ml) or biomass containing 2.5 .mu.g/ml of plantaricin A,
respectively. Data is the average of three triplicate
experiments.
[0033] FIG. 6 shows Caco2/TC7 cell viability measured as Neutral
Red absorption after 24, 48 and 72 hours of incubation with
purified plantaricin A (2.5 .mu.g/ml) produced by Lactobacillus
plantarum DC400 (DC400) mono-culture or with Lactobacillus rossiae
DPPMA174 (DC400-DPPMA174) co-culture. The purified fraction of L.
rossiae DPPMA174 mono-culture eluted according to chromatographic
conditions as for plantaricin A, has been used as negative control
(DPPMA174). Another negative control is DMEM culture medium (DMEM).
Chemically synthesized plantaricin A (2.5 .mu.g/ml) has been used
as positive control (PlnA). Data is the average of three triplicate
experiments. Asterisk indicates significant differences (P<0.01)
with respect to negative control.
[0034] FIG. 7 shows Caco2/TC7 cell viability measured as absorption
of Neutral Red after 24, 48 and 72 hours of incubation with
.gamma.-interpheron (IFN-.gamma.) (1000 U/ml) alone and with
IFN-.gamma.+purified plantaricin A (2.5 .mu.g/ml)
(IFN-.gamma.+DC400-DPPMA174). Purified plantaricin is from
Lactobacillus plantarum DC400 and Lactobacillus rossiae DPPMA174
co-culture. DMEM medium culture has been used as negative control
(DMEM). Chemically synthesized plantaricin A (2.5 .mu.g/ml)
together with IFN-.gamma. has been used as positive control (PlnA).
Data is the average of three triplicate experiments. Asterisk
indicates significant differences (P<0.01) with respect to
negative control.
[0035] FIG. 8 shows Transepithelial Electric Resistance (TEER)
(Ohms.times.cm.sup.2) of Caco2/TC7 cells after 24 and 48 hours of
incubation. The incubation has been carried out with: purified
plantaricin A (2.5 .mu.g/ml) from Lactobacillus plantarum DC400
(DC400) mono-culture; purified plantaricin A (2.5 .mu.g/ml) from L.
plantarum DC400 and Lactobacillus rossiae DPPMA174 (DC400-DPPMA174)
co-culture; chemically synthesized plantaricin A (2.5 .mu.g/ml)
(PlnA); .gamma.-interpheron (IFN-.gamma.) (1000 U/ml) and purified
plantaricin A from L. plantarum DC400 and L. rossiae DPPMA174
(IFN-.gamma.+DC400-DPPMA174) co-culture; and IFN-.gamma. and
chemically synthesized plantaricin A (PlnA+IFN-.gamma.). DMEM
culture medium (DMEM) has been used as negative control. Data is
the average of three triplicate experiments. Asterisk indicates
significant differences (P<0.01) with respect to negative
control.
[0036] FIG. 9 shows results of wound healing assays on
keratinocytes treated with the control, plantaricin A or
plantaricin A containing biomass, respectively.
[0037] FIG. 10 shows the distance between the margins of a wound
treated with control, plantaricin A or plantaricin A containing
biomass.
[0038] FIG. 11 shows increment percentage of healing for the wound
treated with the control, plantaricin A or plantaricin A containing
biomass.
[0039] FIG. 12 shows representative images of human 2544 NCTC
keratinocyte monolayer treated, following the cut, with co-cultured
Plantaricin (a) and synthetic Plantaricin (b), at considered
different time intervals. The monolayer cut has been carried out
using 200 .mu.l pipette tip. The cells have been treated with
co-cultured and synthetic Plantaricin independently and at
concentrations of 0.1, 1 and 10 .mu.g/ml.
[0040] FIG. 13 shows the percentage of cells migrated through the
cut area after treatment with co-cultured Plantaricin (0.1, 1 and
10 .mu.g/ml). The migration percentages have been determined
measuring the clear area between the two cut lines at 0, 4, 8, 12,
24, 48 and 72 hours after the cut. Bars represent average.+-.S.E.M
of two triplicate independent experiments.
[0041] FIG. 14 shows the percentage of cells migrated through the
cut area at starting time after treatment with synthetic
Plantaricin (0.1, 1 and 10 .mu.g/ml). The migration percentages
have been determined measuring the clear area between the two cut
lines at 0, 4, 8, 12, 24, 48 and 72 hours after the cut compared to
initial clear area. Bars represent average.+-.S.E.M of two
triplicate independent experiments.
[0042] FIG. 15 shows the percentage of clear area with respect to
initial cut area after treatment of human NCTC2544 keratinocyte
monolayer with co-cultured Plantaricin (0.1, 1 and 10 .mu.g/ml).
The migration percentages have been determined measuring the clear
area between the two cut lines at 0, 4, 8, 12, 24, 48 and 72 hours
after the cut compared to initial clear area. Bars represent
average.+-.S.E.M of two triplicate independent experiments.
[0043] FIG. 16 shows the percentage of clear area with respect to
initial cut area after treatment of human NCTC2544 keratinocyte
monolayer with synthetic Plantaricin (0.1, 1 and 10 .mu.g/ml). The
percentages have been determined measuring the clear area between
the two cut lines at 0, 4, 8, 12, 24, 48 and 72 hours after the cut
compared to initial clear area. Bars represent average.+-.S.E.M of
two triplicate independent experiments.
[0044] FIG. 17 shows the effect of co-cultured Plantaricin (0.1, 1
and 10 .mu.g/ml), synthetic Plantaricin (0.1, 1 and 10 .mu.g/ml)
and Connectivine (200 .mu.g/ml) on TGF.beta.1 expression in human
NCTC 2544 keratinocyte monolayer, incised in order to have a 200
.mu.l pipette tip cut.
EXAMPLE 1
Synthesis and Purification of Plantaricin Type Peptide Pheromones
and Study of the Effects Thereof on Epidermis and Caco-2 Cells
[0045] L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM)
from the Collezione di Colture del Dipartimento di Protezione delle
Piante e Microbiologia Applicata dell'Universita degil Studi di
Bari, previously isolated from "natural sourdoughs", have been
propagated at 30.degree. C. for 24 hours in modified MRS media
(mMRS), containing, in addition to usual ingredients, 5% maltose
and 10% sourdough water--final pH 5.6.
[0046] 24 h cultured cells, collected by centrifugation
(10.000.times.g for 15 min at 4.degree. C.), washed twice with 50
mM phosphate buffer, pH 7.0 and re-suspended in water at cell
density of 9.0 log ufc/ml have been inoculated (4%, for each
species) in mono- or co-culture conditions on WFH culture media
(Gobbetti, 1998. The sourdough microflora: interactions of lactic
acid bacteria and sourdoughs. Trends Food Ski Technol 9:267-274).
Same procedure has been applied and same results, successively
described, have been obtained using grape must (diluted at 1% of
soluble carbohydrates, added with 0.5% maltose and 0.5% sourdough
extract, pH 5.6), milk serum (see previous integrations) or aqueous
extracts of vegetable and fruit products (see previous
integrations) as culture substrates. The incubation is carried out
for 18 hours at 30.degree. C. After cell removal by centrifugation
(10.000.times.g for 10 min at 4.degree. C.), mono- and co-culture
supernatants have been added with trifiuoroacetic acid (0.05%) and
centrifuged at 10.000.times.g for 10 min. The supernatant has been
filtered using 0.22 .mu.m pore filters. HPLC analysis has been
carried out using AKTA Purifier (GE Healthcare) apparatus equipped
with a detector operating at 214 nm and using a reverse phase C18
XTerra column (Waters, Mildford). Mixture of water, 2-propanol and
trifluoroacetic acid (0.05%) has been used as mobile phase. All
type A plantaricin containing fractions have been analyzed using
ESI-ion trap MS mass spectrometer coupled multidimensional
chromatograph (MDLC). The analysis conditions for the
identification and quantification of plantaricins A, K and N are
according to Di Cagno et al. (Di Cagno et al., 2010. Quorum sensing
in sourdough Lactobacillus plantarum DC400 (DSM 23213): induction
of plantaricinA (PlnA) under co-cultivation with other lactic acid
bacteria and effect of PlnA on bacterial and Caco-2 cells.
Proteomics in press).
[0047] (2) Growth Kinetics of Lactobacillus Rossiae DPPMA174 (DSM
23214)
[0048] Growth kinetic data of L. rossiae DPPMA174 (DSM 23214) have
been processed using Gompertz equation, successively modified
(Zwietering et al., 1990. Modelling of bacterial growth curve. Appl
Environ Microbiol 56: 1875-1881). Cell counting has been carried
out by plating on SDB culture medium at 30.degree. C. for 48 hours.
Cell viability and number of damaged and/or died cells have been
determined by means of LIVE/DEAD bacLight Bacterial Viability kit
(Molecular Probes, INc., Cambridge).
[0049] (3) Two-Dimensional Electrophoretic Analysis and Proteins
Identification
[0050] Two-dimensional electrophoretic analysis of cytoplasmic
proteins from mono- or co-cultured L. rossiae DPPMA174 (DSM 23214)
has been carried out using immobiline-polyacrylamide system (De
Angelis et al., 2005. Biochim. Biophys. Acta. 1762:80-93). Four
gels for each condition have been analysed and the data have been
standardized according to procedure by Bini et al. (Bini et al.,
1997. Protein expression profiles in human breast ductal carcinoma
and histologically normal tissue. Electrophoresis.
18:2831-2841).
[0051] Protein identification has been carried out using
LC-ESI-MS/MS analysis and comparison of obtained sequences to
various databases (National Center for Biotechnology Information,
Bethesda, Md., USA; ProFound,
http://www.prowl.rockefeller.edu/cgibin/ProFound).
[0052] (4) Assays on Reconstructed Epidermis and TEER
(Transepithelial Electric Resistance) Determination
[0053] Reconstructed human epidermis SkinEthic.RTM. (Reconstructed
Human Epidermis) consists of normal kenatinocytes from multi-layer
human epidermis. It is a completely differentiated epidermis from
human keratinocytes culture in a chemically defined medium (MCDM
153), without calf serum addition, on an inert porous polycarbonate
support at air-liquid interface over 17 days. At this growth step
the morphologic analysis shows a multi-layered viable epidermis and
corneous layer consisting of more than ten compact cellular layers.
Reconstructed human epidermis SkinEthic.RTM. has been used
according to previously described protocol (Di Cagno et al., 2009.
Synthesis of .gamma.-amino butyric acid (GABA) by Lactobacillus
plantarum DSMZ19463: functional grape must beverage and
dermatological application. Appl Biotechnol Microbiol DOI:
10.10071s00253-009-23704).
[0054] TEER determination has been carried out using Millicell-ERS
Volthommeter (Di Cagno et al., 2010. Quorum sensing in sourdough
Lactobacillus plantarum DC400: induction of plantaricin A (PlnA)
under co-cultivation with other lactic acid bacteria and effect of
PlnA on bacterial and Caco-2 cells. Proteomics in press).
[0055] (5) Assays on Caco-2/TC7 Cells
[0056] Human Caco-2/TC7 cells (TC7 clone) have been cultured in
Dulbecco medium (DMEM), added with calf serum (10%), not essential
amino acids (1%), gentamycin/streptomycin (50 .mu.g/ml), glutamine
(2 mM) and 4-2-hydroxyethyl-1-piperazinil-etansulfonic acid (1%)
(Di Cagno et al., 2010. Quorum sensing in sourdough Lactobacillus
plantarum DC400: induction of plantaricin A (PlnA) under
co-cultivation with other lactic acid bacteria and effect of PlnA
on bacterial and Caco-2 cells. Proteomics in press). Cell viability
has been determined with absorption assay using Neutral Red dye
(Balls et al., 1987. Approaches to validation alternative methods
in toxicology. In: Goldber A. M. (Ed). N.Y. Academic Press pp.
45-58). After 24-72 hour treatment with various preparations, cells
have been washed with PBS buffer and incubated for 4 hours at
37.degree. C. with a Neutral Red solution (33 mg/l). Successively,
the cells have been again washed with PBS buffer and treated with
lysis solution (50% ethanol in 1% acetic acid containing water).
Plate reading has been carried out using Novapath plate reader
(Biorad, Hercules, Calif.). Di Cagno et al., 2010. Quorum sensing
in sourdough Lactobacillus plantarum DC400: induction of
plantaricin A (PlnA) under co-cultivation conditions with other
lactic acid bacteria and effect of PlnA on bacterial and Caco-2
cells. Proteomics in press).
[0057] For TEER determination Caco-2/TC7 cells have been inoculated
(7.5.times.10.sup.4 cells/ml) in a 24 well plate and a polyethylene
filter (0.4 .mu.m pore). Before the treatment, the cells have been
incubated for 21 days at 37.degree. C. Treatments with various
preparations have been carried out for 18, 24 and 48 hours.
Integrity of the cellular layer therefore has been determined by
means of TEER determination.
[0058] Results
[0059] (1) Synthesis and Purification of Plantaricin Type Peptide
Pheromones
[0060] After 18 hour growth in WFH medium culture the cell density
of L. plantarum DC400 (DSM 23213) mono-culture was 9.27.+-.0.18 log
ufc/ml. .mu..sub.max and A values were, respectively, about 0.27
log ufc/ml/h and 3.77 h. The cell density of lactic acid bacteria
changed from 9.0.+-.0.05 (L. brevis CR13) to 9.43.+-.0.31 log
ufc/ml (L. plantarum DPPMA20). .mu..sub.max values varied from
0.11.+-.0.05 (L. pentosus 12H5) to 0.15.+-.0.04 log ufc/ml/h (L.
plantarum DPPMA20), as weel as .lamda. value changed from
0.37.+-.0.07 (P. pentosaceus 2.times.A3) to 4.20.+-.0.36 h (L.
rossiae DPPMA174 (DSM 23214)). In comparison to mono-culture, the
cell density of L. plantarum DC400 (DSM 23213) did non change
significantly (P>0.05) (9.06.+-.0.34-9.28.+-.0.42 log ufc/ml)
when the micro-organism has been co-cultured with the other lactic
acid bacteria. Also cell yield for L. plantarum DPPMA20,
Lactobacillus paralimentarius 8D, L. pentosus 12H5, Lactobacillus
reuteri e Weissella cibaria 10.times.A16 has not been conditioned
by the co-culture conditions. On the contrary, the cell density of
L. rossiae DPPMA174 (DSM 23214) and P. pentosaceus 2.times.A3 is
remarkably decreased (P<0.05) (about 8.08 and 8.39 log cfu/ml)
compared to mono-culture conditions. Generally .mu..sub.max values
are decreased for all the lactic acid bacteria when co-cultured
with L. plantarum DC400 (DSM 23213). With the exception of L.
plantarum DPPMA20, also the .lamda. latency phase is increased for
all the lactic acid bacteria. Lactic acid bacteria strains that
have shown an inhibition (L. rossiae DPPMA174 (DSM 23214) and P.
pentosaceus 2.times.A) as a result L. plantarum DC400 (DSM 23213)
co-culture and some strains not affected by co-culture conditions
(L. plantarum DPPMA20 and L. pentosus 12H5) have been used in
successive experiments.
[0061] In agreement with the previous results, the number of L.
rossiae DPPMA174 (DSM 23214) viable cells is decreased from
9.0.+-.0.28 to 8.32.+-.0.25 log cellule/ml from mono-culture to
co-culture conditions with L. plantarum DC400 (DSM 23213). Number
of viable and culturable cells did not show significant differences
(P>0.05). Again with reference to mono-culture conditions, the
number of dead or damaged L. rossiae DPPMA174 (DSM 23214) cells is
significantly (P<0.05) increased in co-culture conditions with
L. plantarum DC400 (DSM 23213). Also the number of culturable P.
pentosaceus cells is significantly (P<0.05) decreased when the
lactic bacterium has been co-cultured with L. plantarum DC400 (DSM
23213).
[0062] After cell removal, the mono- and co-culture supernatants
are used for the determination of plantaricin type peptide
pheromones using nano-ESI/MS-MS mass spectrometry coupled MDLC
analysis. FIG. 1a shows full-scan chromatogram of L. plantarum
DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214) co-culture
sample. Because of the matrix complexity, it has been possible to
identify some species, while on the contrary it was difficult to
carry out the complete separation of adjacent peaks. However, it
was possible to separate the co-eluted species by signal filtration
in correspondence of particular m/z values. An example of
identified species is reported in FIG. 1b. MS/MS spectra have been
obtained for each species. For example FIG. 1c shows the spectrum
corresponding to 1493.7 m/z value, selected for sample from L.
plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214)
co-culture. For the sequence search on NCBInr database the
following parameters have been specified: genus (Lactobacillus),
m/z tolerance ratio for ion recognition (0.2 Da) and
instrumentation used for analysis. The presence of plantaricin A
(SEQ ID NO:1
Lys-Ser-Ser-Ala-Tyr-Ser-Leu-Gln-Met-Gly-Ala-Thr-Ala-Ile-Lys-Gln-Val-Lys-L-
ys-Leu-Phe-Lys-Lys-Trp-Gly-Trp) has been observed both for L.
plantarum DC400 (DSM 23213) and DPPMA20 mono-cultures and all
co-cultures wherein DC400 strain has been cultured with other
lactic acid bacteria.
[0063] Based on previous results the samples from mono- and
co-cultures have been purified using 4 chromatographic runs and
further analyzed using nano-ESI-MS in order to exclude other
peptide contamination. The concentration of L. plantarum DC400 (DSM
23213) synthesized plantaricin A has been determined by
chromatographic analysis, using a reverse phase C18) (Terra column
(Waters, Mildford). and OPA method. FIG. 2 shows the concentration
of plantaricin A under various conditions. It is possible to
observe that the synthesis yield of the peptide pheromone is
increased from mono-culture (about 0.06 .mu.g/ml) to co-culture
conditions in the presence of L. rossiae DPPMA174 (DSM 23214)
(about 2.5 .mu.g/ml). Under certain experimental conditions the
concentration of plantaricin A is about 4.0 .mu.g/ml. Although
under co-culture conditions with other lactic acid bacteria species
the production of plantaricin A has been observed, the obtained
amount is remarkably lower than for L. plantarum DC400 (DSM 23213)
and L. rossiae DPPMA174 (DSM 23214) co-culture.
[0064] This result proves that the synthesis of the peptide
pheromone is specific in the presence of specific microbial
interactions suitable to induce the release of signal molecules.
The synthesis of plantaricin A starts during the intermediate
exponential growth phase (about 7 hours) and increases up to the
end of the exponential phase (about 12 hours). Although at lower
concentrations, i.e. about 0.02-0.06 .mu.g/ml, also type K and N
plantaricins have been detected only in L. plantarum DC400 (DSM
23213) and L. rossiae DPPMA174 (DSM 23214) co-culture. Same results
have been obtained using CDM, grape must, milk serum or fruit and
vegetable product aqueous extracts as substrate for the co-culture
cultivation.
[0065] (2) Growth Kinetics of Lactobacillus Rossiae DPPMA174 (DSM
23214)
[0066] L. rossiae DPPMA174 (DSM 23214) has been cultivated on WFH
culture medium added with 2.5 .mu.g/ml of purified or chemically
synthesized plantaricin A. In FIG. 3 it is observed that the
presence of purified plantaricin A resulted in a remarkable
decrease of cell number, i.e. from 9.18.+-.0.26 (under mono-culture
conditions) to 8.4.+-.0.14 log ufc/ml. Similar results have been
obtained using chemically synthesized plantaricin A. In both these
cases results similar to those found for L. plantarum DC400 (DSM
23213) and L. rossiae DPPMA174 (DSM 23214) co-culture have been
observed. Damaged or died L. rossiae DPPMA174 (DSM 23214) cell
number when culture has been carried out in the presence of
purified or chemically synthesized plantaricin A has been
significantly (P<0.05) higher than for mono-culture (about
8.80.+-.0.14 vs. 6.08.+-.0.22 log cells/ml).
[0067] Obtained data show that inhibitory effect of L. plantarum
DC400 (DSM 23213) against L. rossiae DPPMA174 (DSM 23214) results
from the synthesis of plantaricin A and, probably, other peptide
pheromones belonging to the same chemical class.
[0068] (3) Variation of the Protein Expression Levels in L. Rossiae
DPPMA174 (DSM 23214)
[0069] In comparison to the mono-culture, the two-dimensional
electrophoretic analysis of cytosol extracts of L. rossiae DPPMA174
(DSM 23214) cultivated in co-culture with L. plantarum DC400 (DSM
23213) or in the presence of purified plantaricin A has shown the
variation of the expression level of 51 and 27 proteins,
respectively. All the hyper-expressed proteins in the presence of
the plantaricin A have been also hyper-expressed also under
co-culture conditions. By way of example, FIG. 4 shows portions of
gels referring to mono-culture, co-culture with L. plantarum DC440
and mono-culture in the presence of purified plantaricin A
conditions. Some of more hyper-expressed proteins have been
identified using mass spectrometry analysis and found to be
involved in protein biosynthesis (seryl-tRNA synthetase), energetic
metabolism (glucose-6-phosphate dehydrogenase, phosphoglycerate
mutase, acetaldehyde-CoA dehydrogenase, 6-phospho-gluconate
dehydrogenase and .beta.-phospho-gluco-mutase), katabolism of
proteins and amino acids
[0070] (ATP-dependent Clp proteinase and R aminopeptidase),
environmental-stress responses (GroEL, GroES, S2 and S5 ribosomial
proteins) and redox potential homeostasis (NADH oxidase). The
majority of these proteins has been identified also in other lactic
acid bacteria as a response to environmental stress conditions
and/or cell communication mechanisms (Di Cagno et al., 2007.
Cell-cell communication in sourdough lactic acid bacteria: a
protomic study in Lactobacillus sanfranciscensis CB1. Proteornics
7:2430-2446). Particularly, glucose-6-phosphate dehydrogenase
enzyme catalyzes the release of a fratricide pentapeptide in the
mechanisms of Escherichia coli cell programmed death (Kolodkin-Gal
et al., 2007. A linear pentapeptide is a quorum-sensing factor
required for mazef-mediated cell death in Escherichia coli. Science
318:652-655).
[0071] The obtained results show that the inhibitory effect of L.
rossiae DPPMA174 (DSM 23214) as a result of plantaricin A synthesis
is based on cell communication mechanism and, probably, is suitable
to trigger responses resulting in target microorganism death. These
result prove a bactericidal valence of the signal molecule.
[0072] (4) Assays on Reconstructed Epidermis and TEER
[0073] (Transepithelial Electric Resistance) determination Both a
biomass sample from Plantarum DC400 (DSM 23213) and L. rossiae
DPPMA174 (DSM 23214) co-culture with pantaricin A concentration of
2.5 .mu.g/ml and co-culture deriving purified plantaricin A at same
concentration of 2.5 .mu.g/ml have been assayed for the treatment
of reconstructed human epidermis according to SkinEthic.RTM. model.
This model has been widely used and accepted by the scientific
community (Di Cagno et al., 2009. Synthesis of .gamma.-amino
butyric acid (GABA) by Lactobacillus plantarum DSMZ19463:
functional grape must beverage and dermatological application. Appl
Biotechnol Microbiol DOI: 10.1007/s00253-009-23704). After 24 hour
treatment TEER measurements have been carried out. This type of
analysis, widely accepted by the international scientific
community, evaluates the tissue corrosion taking as a reference the
integrity of the corneous layer and barrier function. Particularly,
using this evaluation it is possible to obtain information about
the presence of a lamellar compact structure at corneous layer
level, tight integral junctions and epidermal thickness. These
factors as a whole define an efficient barrier function. FIG. 5
shows that both in the presence plantaricin containing biomass and
subject co-culture deriving purified plantaricin A a significant
increment (P<0.05) of TEER value occurs, thus demonstrating a
protective activity of the molecule at cutaneous level. Same result
has been obtained using chemically synthesized plantaricin A.
[0074] According to the current state of the art, this the first
example of application of a peptide pheromone, involved in bacteria
cell communication mechanisms, suitable to be sensed by epidermis
human cells resulting in stimulation of barrier function.
[0075] (5) Assays on Caco-2/TC7 Cells
[0076] Viability of Caco-2/TC7 cells has been evaluated as Neutral
Red dye adsorption ability. In comparison to DMEM medium (negative
control), the incubation for 24-72 hours with purified plantaricin
A (2.5 .mu.g/ml) remarkably increased the viability of Caco-2/TC7
cells (FIG. 6). Same result has been obtained using chemically
synthesized plantaricin A. No induction has been observed with
treatment using sample deriving from L. rossiae DPPMA174 (DSM
23214) mono-culture purified fraction eluted according to same
chromatographic conditions used for plantaricin A. As expected the
exposure of Caco-2/TC7 cells to .gamma.-interpheron (IFN-.gamma.)
resulted in a remarkable viability decrease (P<0.05) (FIG. 7).
On the contrary, the negative effect of IFN-.gamma. is completely
eliminated in the presence of a simultaneous treatment with
purified or chemically synthesized plantaricin A. The addition of
purified plantaricin A resulted in a remarkable increase
(P<0.05) of TEER values for Caco-2/TC7 cells (FIG. 8). The same
result has been observed using Caco-21TC7 cells. In comparison to
DMEM medium, the addition of IFN-.gamma. significantly (P<0.05)
reduced TEER values. However, the addition of purified or
chemically synthesized plantaricin A eliminated also in this case
the negative effects.
[0077] Caco-2/TC7 cells are one of in vitro most used system in
order to simulate the intestinal mucosa. Although neoplastic origin
thereof, said cells are suitable to differentiate spontaneously in
mature enterocytes and express brush border enzymes. Under culture
conditions, Caco-2/TC7 cells are suitable to develop morphological
and functional characteristics, including tight intercellular
junctions, whose integrity is determined by TEER determinations
(Sambuy et al., 2005. The Caco-2 cell line as a model of the
intestinal barrier: influence of cell and culture-related factors
on Cao-2 cell functional characteristics. Cell Biol Toxicol
21:1-26). The results of this study demonstrate that purified L.
plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214)
co-culture deriving or chemically synthesized plantaricin A is
suitable to stimulate the barrier function of intestinal mucosa and
prevent negative effects of .gamma.-interpheron treatments.
[0078] (6) Development of a Biotechnological Protocol for the
Synthesis of Plantaricin a and Use Thereof in Dermatological
Field
[0079] As above outlined in other part of the text, a
biotechnological process for the synthesis of plantaricin A and use
thereof in dermatological field has been developed. Said process
comprises:
[0080] a) Cultivation of L. plantarum DC400 (DSM 23213) and L.
rossiae DPPMA174 (DSM 23214) in pure culture on mMRS culture
medium;
[0081] b) Cell collection, washing, re-suspension in WFH, CDM,
grape must, milk serum or vegetable or fruit aqueous extracts
suitably integrating for nutrient availability as culture
medium;
[0082] c) culture incubation for 18-24 hours, preferably for 24
hours at 30-37.degree. C., preferably 30.degree. C.;
[0083] d) cell separation by centrifugation. According to a process
variant the preparation can also contain lactic acid bacteria
cells;
[0084] e) preparation dehydration by drying or freeze-drying
process;
[0085] f) production of dermatological preparation.
EXAMPLE 2
Study about the Effect of the Biomass According to the Invention
and Plantaricin a in Wound Healing
[0086] Method
[0087] Cultured human keratinocytes have been incubated with
Plantaricin
[0088] A (2.5 .mu.g/ml) or Plantaricin A containing biomass for 1
hour.
[0089] At the end of the incubation, the cells have been washed and
culture medium restored.
[0090] Wound Healing
[0091] The test consists of carrying out a mechanical interruption
in the continuity of the cellular monolayer in order the treatment
effect in favouring or not the keratinocyte ability to migrate
beyond the damage border and therefore "heal the lesion" to be
evaluated.
[0092] To this end the keratinocyte monolayer, treated as above
described, was incubated over aditional 24 hours after stimulus
application and then fixed and stained using ematoxylin/eosin
staining. Images have been observed with light microscope using
5.times. objective.
[0093] For each image maximum cell migration limits and distance or
gap there between have been detected and calculated, respectively
(FIG. 9-10).
[0094] Results
[0095] In FIG. 9, results of wound healing assay are reported.
After a 24 hour incubation following the interruption of cellular
continuity, not stimulated keratinocyte monolayer (control)
displays some cells extending from wound margins. The cellular
migration is however particularly apparent when the cells are
stimulated with hyaluronic acid. In this case in fact the lesion
gap is narrower than for not treated cells (control), evidencing
the cell migration ability for wound healing.
[0096] In order to estimate analytically the cell migration
ability, the wound margins have been outlined, measured and
analyzed as reported in FIG. 10.
[0097] Plantaricin A or Plantaricin A containing biomass incubated
keratinocytes reduce the gap between the margins wound in
comparison to control cell, the percentage of healing increase in
comparison to control is reported in FIG. 11.
[0098] Assay System: Human Reconstructed Epidermis
[0099] Used epidermis model is produced by Skinethic.RTM.
Laboratories, Nice (France) and is used as 0.5 cm.sup.2 specimen
from differentiation 17.sup.th day with batch average thickness of
120.mu.. (corneous layer and vital epidermis).
[0100] Completely differentiated epidermis is obtained from human
keratinocytes cultivated in a chemically defined medium (MCDB 153)
without calf serum addition, on porous polycarbonate inert support
at air-liquid interface over 17 days; at this differentiation stage
the morphologic analysis shows a multilayer vital epidermis and
corneous layer consisting of more than 10 compact cell layers.
[0101] TEER Determination
[0102] Trans-epithelial electric resistance (TEER) is a direct
measurement of the cutaneous barrier functionality: it reflects the
tissue resistance as a whole resulting from both thickness and
structure. It reflects integrity of the intercellular contacts at
tight junction level, bi-lamellar lipid structure protecting from
the penetration of outside substances.
[0103] TEER is assay discriminating parameter--Rat skin electrical
resistance (B 40)--EU validated test for the corrosiveness
evaluation considering as end-points the integrity of the corneous
layer and barrier function.
[0104] It is inversely proportional to TEWL as in vivo measured,
which is the measure of trans-epidermal water loss: higher TEWL
corresponds to higher damage of barrier function while higher TEER
corresponds to lower damage of barrier function.
[0105] Over the insert 1 ml of PBS is dosed and the
trans-epithelial electric resistance is measured using
Millicell-ERS instrument (range 0-20 k.OMEGA.).
[0106] Various measurements have been carried out for each
tissue.
[0107] FIG. 5 shows TEER values referring to an average for 3
tissues, 3 determinations being carried out for each thereof.
[0108] In relation to TEER value the following properties are
important: the presence of a lamellar compact structure at corneous
layer level, integral tight junctions and epidermal thickness that
as a whole define an efficient barrier function. Every tissue is
own reference with determinations at t=0 and t=24 hours.
[0109] Obtained results are particularly interesting, in fact it is
apparent a TEER remarkable increase both in the presence of
Plantaricin A and Plantaricin A containing biomass.
[0110] This increase is a directed consequence both of the
epidermal thickness increase and a better compactness and integrity
of the corneous layer at tight junctions level.
EXAMPLE 3
Plantaricin A
Study about the Role Thereof for the Maintenance and Restoration of
Cutaneous Barrier Function
[0111] The study is based on the use of Plantaricin A obtained by
L. plantarum DC400 (DSM 23213) and L. rossiae DPPMA174 (DSM 23214)
co-culture in order the following goals to be obtained:
[0112] Evaluation of the biomass effects on wound repair, through
the study of the effects on the migration and proliferation of
human keratinocyte monolayer (NCTC2544) compared to a positive
control (not treated cells) and a commercially available/negative
known activity control. The treatments will be carried out at
different concentrations of the subject Plantaricin (after
determination using cytotoxicity assay) at three successive times
(24, 48, 72 hours).
[0113] Analysis of the cell damage response, at considered times
and concentrations, evaluating the mediator modulation like IL-8,
KGF (keratinocyte growth factor), TGF-.beta.1 (transforming growth
factor-.beta.), compared to a positive control (not treated cells)
and a commercially available/negative known activity control, by
means of Real-Time PCR.
[0114] Materials and Methods
[0115] Cell Cultures
[0116] The used cell line is a human NCTC 2544 keratinocyte cell
line (Perry, V. P., Sanford, K. K., Evans, V. J., Hyatt, G. W.,
Earle, W. R., 1957. Establishment of clones of epithelial cells
from human skin. J Natl Cancer Inst. 18 (5): 709-717) cultured in
sterile flasks, incubated at 37.degree. C. in humid atmosphere at
5% CO.sub.2 in MEM (Minimum Essential Medium) culture medium added
with 10% bovine calf serum (FBS), 2 mM L-glutamine, 1% not
essential amino acids, in the presence of 1% penicillin and
streptomycin. Cells grow in vitro adhering to culture plate surface
as a monolayer.
Study on the Tissue Damage Remedy Over Time by Means of Wound
Healing
Principle of the Method:
[0117] The experiment firstly involves the development of confluent
cell monolayer. Successively the cut is carried out and the
generated "gap" is observed using microscope as cells gradually
moving repair the damage.
[0118] This cicatrisation process (said "healing") can last from
several hours to more than a day depending on cell line, wound
conditions and extent.
[0119] Experimental Procedure
[0120] NCTC 2544 cells are plated and cultured at 37.degree. C.,
with 5% CO.sub.2 for 24 hours.
[0121] After 24 hours complete growth medium is replaced with serum
free medium to avoid the serum effect on cell proliferation and
cells are incubated for further 24 hours at 37.degree. C., 5%
CO.sub.2.
[0122] After 24 hours the cell monolayer is mechanically damaged by
mild 200 .mu.l pipette tip brushing tracking a .about.1 mm wide
horizontal line. Then the monolayer is washed and after addition of
substances to be tested, the plates are incubated at 37.degree. C.,
5% CO.sub.2 for 72 hours.
[0123] the effect of the substances on cell motility is evaluated
using phase contrast reverse microscope, acquiring images at
various opportunely selected analysis times.
[0124] the damage remedy is determined measuring the clear area
between the cut two migration fronts at 0, 4, 8, 12, 24, 48, 72
hours after the cut, using an image processing software (Leica
application Suite).
[0125] All the data for each experiment are Excel statistically
processed for determinations at 0, 4, 8, 12, 24, 48 and 72
hours.
Analysis of Cell Damage Response by Evaluation of IL-8, KGF,
TGF-.beta. Gene Expression Using Real-Time PCR
[0126] The procedure consists of 3 fundamental steps: I. Extraction
of total RNA from the cells II. Retro-transcription in cDNA
III. Real-Time PCR
[0127] I. Extraction of Total RNA from the Cells
[0128] Immortalized NCTC 2544 human keratinocyte cell line,
maintained in culture flasks, incubated at 37.degree. C. in humid
atmosphere at 5% CO.sub.2 in MEM (Minimum Essential Medium) culture
medium added with 10% calf serum (FBS), 2 mM glutamine, 1% not
essential amino acids, in the presence of 1% penicillin and
streptomycin, is used. The cell line will be scraped using a 200
.mu.l pipette tip and treatments at different times and
concentrations.
II. RNA Retro-Transcription in cDNA
[0129] The process includes amplification of RNA samples extracted
using
[0130] "High Capacity cDNA Reverse Transcription Kit" (Applied
Byosistem).
III. Real-Time PCR
[0131] The process includes cDNA amplification using specific
Taqman Gene assay and "TaqMan Universal PCR Master Mix with
Amperase UNG 2X" kit (Applied Biosystem).
[0132] A relative type quantification to determine a possible
variation of target gene expression with respect to a positive
control (not treated cells) will be used by means of housekeeping
gene for data normalization and data analysis according to
2.sup.-.DELTA..DELTA.Ct method.
[0133] Results
[0134] Wound Healing
[0135] The ability of co-culture deriving Plantaricin to modify the
migration of human NCTC 2544 keratinocytes by wound healing assay
has been evaluated.
[0136] The study concerned also the comparative evaluation of the
effects of synthetic and L. plantarum DC400 and L. rossiae DPPMA174
co-culture deriving Plantaricin.
[0137] After the cut on the cellular monolayer has been carried out
using a 200 .mu.l pipette tip and the cells within the gap between
two cut lines completely removed, said cells are treated with
synthetic and co-culture deriving Plantaricin at the following
concentrations: 0.1-1-10 .mu.g/ml. Also positive and negative
controls (connectivine at 100 .mu.g/ml) have been duplicate
tested.
[0138] Images on the same cut area have been acquired at time 0 and
after 4, 8, 12, 24, 48 and 72 hours, to monitor the cell migration
in the cut area.
[0139] FIGS. 12-16 shows the temporal analysis for the effect of
co-cultured (0.1, 1 and .mu.g/ml) (a) and synthetic Plantaricin
(0.1, 1 and .mu.g/ml) (b) on NCTC 2544 cell line migration.
[0140] FIG. 12 shows the cicatrisation progression
(.DELTA..mu.m/time) at each considered time (at 4 hour interval)
for both tested actives and positive and negative controls.
[0141] During early steps of cellular cicatrisation it is not
possible to detect meaningful differences at time 0 in cell
migration between the controls and tested actives. As it is
apparent in FIG. 12a, co-cultured Plantaricin at concentration of
0.1 and 1 .mu.g/ml is not suitable to accelerate remarkably the
cell migration compared to the control. The effect is already
significant 4 hours after the cut and remains constant and
significant up to 72 hours. Treatments with co-cultured Plantaricin
at a concentration of 10 .mu.g/ml produce instead a remedy of the
tissue damage comparable to positive control, i.e. not treated
cells, for all the considered time intervals.
[0142] A similar experiment carried out with a cell treatment for
the same temporal period at equivalent concentrations of synthetic
Plantaricin proved that also in this case the treatments with
Plantaricin (synthetic) have a greater ability to accelerate the
cell migration within the two cut lines in comparison to the
control negative.
[0143] Particularly, also in this case, during early steps of
cellular cicatrisation it is not possible to detect meaningful
differences at time 0 in cell migration both for two used controls
and two under study actives.
[0144] FIG. 12b shows that synthetic Plantaricin at concentration
of 0.1 and 10 .mu.g/ml, respectively, produces an increase of cell
migration higher than both positive and negative controls, already
4 hours after the cut and remains constant for all time intervals
considered up to 72 hours. Treatments with synthetic Plantaricin at
a concentration of 10 .mu.g/ml produce instead a remedy of the
tissue damage comparable to positive and negative controls. In
order to comprise more completely and analyze the effects of the
treatments with co-cultured and synthetic Plantaricin with respect
to positive and negative controls in all the considered time
intervals, we have analyzed statistically the measurements deriving
from the image analysis.
[0145] FIGS. 13 and 14 report the data as percentage of cells
migrated through the two cut lines in comparison to the positive
control for treatments both with co-cultured (FIG. 13) and
synthetic Plantaricin (FIG. 14), respectively. Also the results are
reported as area percentages for the two cut line and initial
areas, respectively (FIG. 15-16).
[0146] Charts reported in FIGS. 13 and 14 show the data relating to
percentages of cells migrated during the various considered time
intervals for positive and negative controls, co-cultured (0.1-1
and 10 .mu.g/ml) and synthetic Plantaricin (0.1-1 and 10 .mu.g/ml),
respectively. In agreement with the data deriving from image
processing, after first 8 hours of treatment, negative control does
not persist in cut cicatrisation activity and presents a percentage
of migrated cells similar to the positive control.
[0147] As to co-cultured Plantaricin (FIG. 13) in general terms it
is possible to assert that, at all three used treatment
concentrations, it is possible to outline a greater increment of
the cell migration from time 0 up to 72 hour treatment, also at
lowest concentration treatment.
[0148] In particular, treatments with co-cultured Plantaricin at
concentration of 10 .mu.g/ml shows highest migration percentage
values for all considered time intervals except after 72 hour
treatment, but these data are not significant with percentage
values of migrated cells of 165,56%, 138.18%, 134.64%, 118.08%,
139.07% and 149.04%, respectively.
[0149] FIG. 14 shows the percentage of cells migrated after
treatment at various considered time intervals, at equivalents
concentrations of synthetic Plantaricin.
[0150] The treatment with synthetic Plantaricin at a concentration
of 0.1 .mu.g/ml produces the highest percentage values of migrated
cells for all the considered time intervals, with values from the
starting time up to 72 hours of treatment of 158.30%, 115.15%,
124.32%, 141.82%, 141.91% and 128.51%, respectively. The treatment
with synthetic Plantaricin at a concentration of 10 .mu.g/ml
results in a percentage of migrate cells for all the considered
time intervals, compared to a treatment with same active at a
concentration of 0.1 .mu.g/ml, but at the same time results in an
higher increase of the percentage of migrated cells compared to
negative control after 8 up to 72 hours of treatment with a
percentage increase of +4.62%, +20.28%, +14.84% and +7.47%,
respectively. On the contrary the treatment with synthetic
Plantaricin at a concentration of 1 .mu.g/ml produces a greater
increase of the percentage of migrated cells compared to negative
control negative only after 12 and 24 hours of treatment with
percentage values of +9.86% and 15.48%, respectively.
[0151] Although the treatment of cells with the same concentrations
of co-cultured and synthetic Plantaricin does not result in a
percentage increase of migrated cells higher than negative control
at all considered time intervals, the comparison of results from
wound healing assays on human NCTC 2544 keratinocytes treated with
co-cultured Plantaricin with those from synthetic Plantaricin under
same conditions allowed to demonstrate that co-cultured Plantaricin
has a greater effect on cell migration through the generated cut
area compared to the considered negative control. TGF.beta.1 gene
is the most involved in the cicatrisation process.
[0152] Among all times considered for wound healing monitoring the
evaluation of gene expression has been carried out after 8 hours of
treatment as a confirmation of the data obtained from cicatrisation
evaluation.
[0153] The obtained results outline and confirm the remarkable
effect of the connectivine on the cicatrisation.
[0154] As to the data on synthetic Plantaricin is pointed out that
the gene expression is remarkably increased even if is possible to
evidence that at same concentrations (1 e 10 .mu.g/ml) co-cultured
Plantaricin stimulates an higher increase of gene expression, a
further confirmation of the biomass effect.
Sequence CWU 1
1
1126PRTLactobacillus plantarum 1Lys Ser Ser Ala Tyr Ser Leu Gln Met
Gly Ala Thr Ala Ile Lys Gln1 5 10 15Val Lys Lys Leu Phe Lys Lys Trp
Gly Trp 20 25
* * * * *
References